Biologically and clinically relevant proteomic data can only be generated if organ or tissue samples investigated consist of homogeneous cell populations, in which no unwanted cells of different types and/or development stages obscure the results. One of the main problems with the analysis of tissue samples is the heterogeneous nature of the sample. Many different cell types are typically present in tissue biopsies, and in the case of diseased tissue small numbers of abnormal cells may lie within or adjacent to unaffected areas. Thus, the laser capture/microdissection process has been developed to provide a rapid and straightforward method for procuring homogeneous subpopulations of cells or structures for biochemical and molecular biological analyses. However, current proteomic techniques, including two-dimensional polyacrylamide gel electrophoresis, multidimensional liquid chromatography systems, and gel and gel-free isoelectric focusing approaches such as chromatofocusing, immobilized pH membranes, Rotofor, free-flow electrophoresis, and off-gel electrophoresis, are all operated at the preparative-scale and are incompatible with small cell populations collected from microdissection-procured specimens. Our research goal is therefore to integrate the Gemini proteomic platform with tissue microdissection/preparation techniques as a novel biomarker discovery paradigm for enabling comprehensive and comparative survey of protein expression profiles in targeted cell populations isolated from clinical tissue specimens. The obvious progression in the development, evaluation, and validation of proposed biomarker discovery paradigm includes (i) the implementation of high throughput Gemini proteomic platform using a multiplexed nano-reversed-phase liquid chromatography system, (ii) full assessment of Calibrant's unique tissue proteome capabilities toward comparative characterization of protein expression profiles within microdissected tumor cells of glioma malignancies, and (iii) confirmation of discriminate biomarker candidates among different grades of glioma malignancies using Western blot and immunohistochemistry techniques on a large series of additional tissue specimens. In addition to the clinical proteomic technology development and validation, Phase II efforts will also focus on the identification of disease markers, exploration of molecular relationships among different disease states and phenotypes, and a deeper understanding of molecular mechanisms that drive disease progression. PUBLIC HEALTH RELEVANCE: Our research goal is to integrate the Gemini proteomic platform emerging from the Phase I project with tissue microdissection/preparation techniques as a novel biomarker discovery paradigm for enabling comprehensive and comparative survey of protein expression profiles in targeted cell populations isolated from clinical tissue specimens. The greatest expectations for targeted proteomic research using enriched malignant cells from high quality tissue specimens reside in the identification of diagnostic, prognostic, and predictive biological markers in the clinical setting, as well as the discovery and validation of new protein targets in the biopharmaceutical industry.